The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated.

Tail-anchored (TA) proteins possess an N-terminal functional domain and a single transmembrane domain (TMD) at the C-terminus followed by a hydrophilic tail.1 Newly synthesized TA proteins are released from free ribosomes with the C-terminal hydrophobic region inserted into various membranes, such as the endoplasmic reticulum, chloroplast outer envelope, mitochondrial outer membrane and the peroxisomal membrane. 2The functional domain of TA proteins orients to the cytosol and the TMD of TA proteins is inserted into membranes posttranslationally. Sorting of proteins by the C-terminal tail (CT) to their specific intracellular destinations is essential for their functions.3 For instance, overexpression of a C-terminal TMDtruncated AtPAP2 in Arabidopsis abolishes its faster plant growth phenotype. 4 Over 500 proteins in Arabidopsis have been predicted to have TA structures, of which, 130 have had their subcellular localization experimentally confirmed based on either GFP targeting or mass spectrometry.2 Most TA proteins were assigned to the ER and secretory membranes, 27 proteins to mitochondria and 32 proteins to plastids.2,5 These include several isoforms of Tom20 and Tom22 (also known as Tom9) of the mitochondrial outer membrane translocon 6,7 and the GTPase receptors of the outer membrane translocon of plastids, including AtToc33, AtToc34. 5,8Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only plant TA protein shown to be dual-targeted to chloroplasts and mitochondria. 4 It was predicted to carry a putative N-terminal signal peptide, a phosphatase domain and a transmembrane domain (TMD) followed by a short hydrophilic C-terminal tail (CT) (a.a. 614-636) by the TMHMM analysis.4 AtPAP2 was to date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (om) of chloroplasts and mitochondria and exposed its n-terminal enzymatic domain to the cytosol. it was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria Keywords: purple acid phosphatase, mitochondria, plastid, dual-targeting, outer membrane detected in the membrane fraction using immunoblotting. 4 An in vivo targeting assay using chimeric GFP vectors showed that the C-terminal TMD motif of AtPAP2, but not the predicted N-terminal signal peptide, can direct GFP to both plastids and mitochondria in Arabidopsis PSB-D protoplasts. 4 In transgenic Arabi...

No abstract

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin–Benson–Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.